level photosynthetic capacity in lowland Amazonian and high‐elevation Andean tropical

17 Pages·2016·1 MB·English

Research
Leaf-level photosynthetic capacity in lowland Amazonian and
high-elevation Andean tropical moist forests of Peru
NurH.A.Bahar1,F.YokoIshida2,LasanthaK.Weerasinghe1,3,RossellaGuerrieri4,5,OdhranS.O’Sullivan1,
KeithJ.Bloomfield1,GregoryP.Asner6,RobertaE.Martin6,JonLloyd2,7,YadvinderMalhi8,OliverL.Phillips9,
PatrickMeir1,5,NormaSalinas8,10,EricG.Cosio10,TomasF.Domingues11,CarlosA.Quesada12,FelipeSinca6,
AlbertoEscuderoVega10,PaolaP.ZuloagaCcorimanya13,JhondelAguila-Pasquel14,15,KatherineQuispe
Huaypar13,IsraelCubaTorres13,RosalbinaButro(cid:1)nLoayza16,YulinaPelaezTapia13,JuditHuamanOvalle13,
BenedictM.Long1,17,JohnR.Evans1,17andOwenK.Atkin1,18
1DivisionofPlantSciences,ResearchSchoolofBiology,TheAustralianNationalUniversity,Canberra,ACT2601,Australia;2CentreforTropicalEnvironmentalandSustainabilityScience,
CollegeofMarineandEnvironmentalSciences,JamesCookUniversity,Cairns,Qld,Australia;3FacultyofAgriculture,UniversityofPeradeniya,Peradeniya20400,SriLanka;4Centrefor
EcologicalResearchandForestryApplications(CREAF),UniversidadAutonomadeBarcelona,EdificioC,08290Cerdanyola,BarcelonaSpain;5SchoolofGeosciences,Universityof
Edinburgh,Edinburgh,EH93JN,UK;6DepartmentofGlobalEcology,CarnegieInstitutionforScience,Stanford,CA94305,USA;7DepartmentofLifeSciences,ImperialCollegeLondon,
SilwoodParkCampus,Ascot,SL57PY,UK;8EnvironmentalChangeInstitute,SchoolofGeographyandtheEnvironment,UniversityofOxford,SouthParksRoad,Oxford,OX13QY,UK;
9SchoolofGeography,UniversityofLeeds,WoodhouseLane,Leeds,LS92JT,UK;10SeccionQuimica,PontificiaUniversidadCat(cid:1)olicadelPeru(cid:1),AvUniversitaria1801,SanMiguel,Lima,
Per(cid:1)u;11FaculdadedeFilosofiaCi^enciaseLetrasdeRibeir~aoPreto,UniversidadedeS~aoPaulo,SaoPaulo,Brazil;12InstitutoNacionaldePesquisasdaAmazonia(INPA),Manaus,Brazil;
13EscuelaProfesionaldeBiologia,UniversidadNacionaldeSanAntonioAbaddelCusco,AvdelaCultura,No.733,Cusco,Peru(cid:1);14InstitutodeInvestigacionesdelaAmazoniaPeruana(IIAP),
Av.Jos(cid:1)eA.Quiñoneskm.2.5,ApartadoPostal784,Iquitos,Peru(cid:1);15SchoolofForestResourcesandEnvironmentalScience,MichiganTechnologicalUniversity,1400TownsendDrive,
Houghton,MI49931,USA;16MuseodeHistoriaNatural,UniversidadNacionaldeSanAntonioAbaddelCusco,AvdelaCultura,No.733,Cusco,Peru(cid:1);17ARCCentreofExcellencefor
TranslationalPhotosynthesis,ResearchSchoolofBiology,TheAustralianNationalUniversity,Building134,Canberra,ACT2601,Australia;18ARCCentreofExcellenceinPlantEnergy
Biology,ResearchSchoolofBiology,TheAustralianNationalUniversity,Building134,Canberra,ACT2601,Australia
Summary
Authorforcorrespondence: (cid:1) Weexaminedwhethervariationsinphotosyntheticcapacityarelinkedtovariationsinthe
OwenK.Atkin environmentand/orassociatedleaftraitsfortropicalmoistforests(TMFs)intheAndes/west-
Tel:[phone]
ernAmazonregionsofPeru.
Email:[email protected]
(cid:1) Wecomparedphotosynthetic capacity (maximal rateof carboxylation of Rubisco (V ),
cmax
Received:3November2015
andthemaximumrateofelectrontransport(J )),leafmass,nitrogen(N)andphosphorus
Accepted:23May2016 max
(P) per unit leaf area (M , N and P , respectively), and chlorophyll from 210 species at 18
a a a
fieldsitesalonga3300-melevationgradient.Westernblotswereusedtoquantifytheabun-
NewPhytologist(2016) danceoftheCO2-fixingenzymeRubisco.
doi:10.1111/nph.14079 (cid:1) Area- and N-based rates of photosynthetic capacity at 25°C were higher in upland than
lowland TMFs, underpinned by greater investment of N in photosynthesis in high-elevation
trees. Soil [P] and leaf P were key explanatory factors for models of area-based V and
Keywords: carboxylationcapacity, a cmax
elevation,leaftraits,nitrogen(N), Jmax but did not account for variations in photosynthetic N-use efficiency. At any given Na
phosphorus(P),ribulosebisphosphate and P , the fraction of N allocated to photosynthesis was higher in upland than lowland
a
regeneration,temperature,tropicalforests. species.ForasmallsubsetoflowlandTMFtreesexamined,asubstantialfractionofRubisco
wasinactive.
(cid:1) These results highlight the importance of soil- and leaf-P in defining the photosynthetic
capacityofTMFs,withvariationsinNallocationandRubiscoactivationstatefurtherinfluenc-
ingphotosyntheticratesandN-useefficiencyofthesecriticallyimportantforests.
prerequisite for modelling carbon storage in tropical ecosystems,
Introduction
withAbeinginfluencedinteraliabynutrientsupply[particularly
Tropicalmoistforests(TMFs)playasignificantroleintheterres- nitrogen(N)andphosphorus(P)],elevationandgrowthtemper-
trialcarboncycle,contributingone-thirdofglobalgrossprimary ature.
productivity(Beeretal.,2010;Malhi,2010).Understandingthe EarlystudiesinlowlandTMFsimplicatedlowfoliarPconcen-
factors that regulate leaf photosynthesis (A) in TMFs is a trationsasamajorinfluenceonlight-saturatednetphotosynthesis
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2 Research Phytologist
(A )(Reich&Walters,1994;Raaimakersetal.,1995),withsoil etal., 1989; Loustau etal., 1999) and reduced N allocation to
sat
P being a major factor limiting Amazon productivity (Quesada Rubisco (Warren & Adams, 2002) under P-limited condi-
etal.,2012).FoliarPiscrucialtothefine-tuningofA (Fredeen tions. Increased allocation of N to nonphotosynthetic compo-
sat
etal.,1989;Jacob&Lawlor,1993)viaregulationofkeyinterme- nents may also play a role (Domingues etal., 2010; Lloyd
diatesincarbonmetabolism(e.g.ATP,NADPHandsugarphos- etal., 2013), as might inactivation of Rubisco (Stitt &
phates including ribulose 1,5-bisphosphate (RuBP)). While the Schulze, 1994). Yet, doubt remains regarding the general
direct effect of P limitation is primarily on RuBP regeneration, V ↔[N] relationship in TMFs because of the scarcity of
cmax
reductionsinRubiscoactivityalsooccur(Brooks,1986;Jacob& data, in both lowland and upland TMFs. Comprehensive sur-
Lawlor, 1992; Loustau etal., 1999). Although Meir etal. (2002, veys of V (and the maximum rate of electron transport
cmax
2007) and Reich etal. (2009) showed that A at a given leaf N (J )) across lowland and upland TMFs are required to estab-
sat max
concentration ([N]) was lower in lowland tropical trees than in lish whether there are generalized patterns of photosynthetic
their temperate counterparts, the extent to which P limitations capacity in relation to environmental conditions and/or other
per se alter A ↔[N] relations within TMFs is uncertain leaf traits.
sat
(Bloomfield etal., 2014a; Domingues etal., 2015). A further TMF species with higher leaf nutrient concentrations and
unknown is the extent to which large elevation gradients affect lower leaf mass per unit leaf area (M ) values are often found in
a
A ↔[N]relationsinthetropics.UplandTMFsaremorelikely more fertile soils (Fyllas etal., 2009), and M tends to increase
sat a
tobelimitedbyNthantheirlowlandcounterparts(Tanneretal., with increasing elevation (Hikosaka etal., 2002; van de Weg
1998). Upland TMFs also experience lower temperatures and etal.,2009;Almeidaetal.,2012;Asneretal.,2014b);leafchem-
atmospheric CO partial pressures, more frequent cloud cover istry also systematically shifts along elevation gradients in the
2
andgreaterleafwetness(Grubb,1977;Vitousek,1984;Girardin tropics (Asner etal., 2014b). Large variations in leaf traits have
etal., 2010; Bruijnzeel etal., 2011). Such factors can limit A also been observed among co-occurring species, reflecting the
sat
(Terashima etal., 1995; Bruijnzeel & Veneklaas, 1998; Letts & importanceofphylogeneticrelationshipsindeterminingtraitval-
Mulligan, 2005), leading to declines in productivity (Girardin ues in TMFs (Townsend etal., 2007; Kraft etal., 2008; Fyllas
etal.,2010).A valuesin uplandTMFs havebeen documented etal.,2009).WhethersimilarpatternsholdforestimatesofV
sat cmax
(e.g. Quilici & Medina, 1998; Cordell etal., 1999; Hikosaka in lowland and upland TMFs (and V ), is, however, not
cmax,N
etal.,2002;Letts&Mulligan,2005;Radaetal.,2009),showing known.
A to be constant with increasing elevation (Cordell etal., Variations in V underlie variations in photosynthetic
sat cmax,N
1999), or declining with increasing elevation (Hikosaka etal., N-use efficiency. Further insights can be gained by quantifying
2002;Wittichetal.,2012). theproportionofNallocatedtothepigment–proteincomplexes
Rates of A are subject to variations in stomatal conduc- (n ), electron transport (n ) and Rubisco (n ) (Evans &
sat P E R
tance (g) and the partial pressure of internal leaf CO (C) Seemann, 1989; Pons etal., 1994; Hikosaka, 2004). Quantifica-
s 2 i
(Santiago & Mulkey, 2003). As variations in C alter both tion of V , J , leaf chlorophyll and [N] can be used to esti-
i cmax max
CO uptake and photorespiratory CO release, variations in mate n , n and n (Evans & Seemann, 1989; Niinemets &
2 2 P E R
C could potentially confound our understanding of how envi- Tenhunen, 1997). In nontropical plants, lower A at a given N
i sat
ronmental gradients alter N investment in A. By contrast, (A )isassociatedwithreducedallocationofNtophotosynthesis
N
variations in g have less impact on the fundamental, biochem- and increased allocation to nonphotosynthetic components
s
ical parameter of photosynthetic capacity – that being the (Poorter & Evans, 1998; Westbeek etal., 1999; Warren &
maximum rate of carboxylation by Rubisco (i.e. V ). Posi- Adams, 2001; Takashima etal., 2004; Hikosaka & Shigeno,
cmax
tive correlations between V and leaf [N] have been 2009). Similarly, variations in A were associated with differ-
cmax N
reported for some tropical species (Carswell etal., 2000; Meir encesinNallocationtoandwithinthephotosyntheticapparatus
etal., 2002, 2007; Domingues etal., 2005; Kumagai etal., in glasshouse-grown tropical tree seedlings (Coste etal., 2005)
2006; V(cid:3)arhammar etal., 2015), whereas in others no strong and in high-elevation TMFs of Rwanda (Dusenge etal., 2015).
V ↔[N] relationship was observed (Coste etal., 2005; van Toourknowledge,nostudyhasquantifiedNallocationpatterns
cmax
de Weg etal., 2012; Dusenge etal., 2015). Although reports infield-growntropicaltrees,andnotwithrespecttofieldsitesin
on V are less widespread in the tropics than those on A , uplandandlowlandTMFs.
cmax sat
the available data suggest that V values, as well as V We examined variations in photosynthetic capacity and leaf
cmax cmax
per unit N (herein termed ‘V ’), are lower in lowland traitsacrossTMFcanopieslocatedat18sitesalonga3300-mele-
cmax,N
TMFs than in their nontropical counterparts (Carswell etal., vationgradientstretchingfromlowlandwesternAmazoniatothe
2000; Meir etal., 2002, 2007; Domingues etal., 2007, 2010; AndeantreelineinPeru.Thestudyincluded11lowlandsitesin
Walker etal., 2014; V(cid:3)arhammar etal., 2015). Kattge etal. northernandsouthernPeru(elevation117–223mabovesealevel
(2009) re-analysed data to show that V per unit N in (asl)),andsevenuplandsitesatelevationsof1527–3379maslin
cmax
TMFs growing on young, relatively high nutrient status soils southern Peru. Our site selection enabled an assessment of the
was higher compared with their older, Ferralsol and Acrisol potential role of P availability on photosynthetic performance
soil counterparts which are characterized by very low soil P acrossAmazonian(cid:3)AndeanTMFsitesdiffering>40-foldintotal
availability (Quesada etal., 2010). These observations are con- soil P. The upland sites were characterized by a floristically dis-
sistent with laboratory studies showing reduced V (Lauer tinct assemblage of montane forest species, with the transition
cmax
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fromlowlandmoistforeststouplandmontaneforestscoinciding cut under water immediately after harvesting to preserve xylem
with an increase in cloud cover (van de Weg etal., 2009; Brui- watercontinuity).
jnzeeletal.,2011).Inconjunctionwiththerecentfindingsofthe CO response curves of light-saturated photosynthesis
2
key role of P in modulating carbon investment (Quesada etal., (A↔C curves) (at 1800lmolphotonsm(cid:3)2s(cid:3)1) were obtained
i
2012) and photosynthesis (Bloomfield etal., 2014b) of tropical within30–60minafterbranchdetachment.CO concentrations
2
trees,andthatleafPvariespredictablyalongsoilPandelevation inside the reference chamber ranged in a stepped sequence from
gradients (Asner etal., 2014b), we addressed the following ques- 35to2000lmolmol(cid:3)1(seeMethodsS2fordetails).Blocktem-
tions: do tropical TMF species growing on low-P soils exhibit peratures within the chamber were set to the prevailing daytime
lower photosynthetic capacity and photosynthetic N-use effi- air temperature at each site (from 25 to 28°C). The resultant
ciency than TMF trees growing on sites with higher P availabil- A↔C curves (examples shown in Fig.1) were fitted following
i
ity? Are there marked differences in V , J and V the model described by Farquhar etal. (1980) in order to calcu-
cmax max cmax,N
betweenlowlandAmazoniananduplandAndeanTMFs?Aredif- lateV andJ onaleafareabasis;seeMethodsS2fordetails.
cmax max
ferences in V , J and V linked to concomitant varia- ForeveryA↔C curve,recordedairpressurewasusedtocorrect
cmax max cmax,N i
tionsinotherleaftraitsand/orenvironmentalvariables? for altitudinal changes in O partial pressure, and to calculate
2
intercellularCO (C)valuesonapartialpressurebasis.
2 i
Rates of CO exchange were corrected for possible gas dif-
MaterialsandMethods 2
fusion through the gasket of the Li-Cor 6400XT leaf chamber
(Bruhn etal., 2002) before calculation of V and J .
Studysites cmax max
Assuming infinite internal diffusion conductance (g ),
m
Fieldworkwascarriedoutin18one-hectarelong-termmonitor- Michaelis constants of Rubisco for CO (K ) and O (K ) at
2 c 2 o
ing plots in Peru which contribute to the ABERG and a reference temperature of 25°C were assumed to be 40.4Pa
RAINFOR networks of permanent sample plots. The plots are and 24.8kPa, respectively (von Caemmerer etal., 1994); these
arrayed along gradients of elevation (117–3379m asl) and soil values were adjusted to actual leaf temperatures assuming acti-
nutrient status (Table1). For each site, climate data were vation energies of 59.4 and 36kJmol(cid:3)1 for K and K ,
c o
obtained from Asner etal. (2014a) and Y. Malhi (unpublished). respectively (Farquhar etal., 1980). Fitted parameters were
Markedchangesinspeciesrichness,canopycoverandtreeheight then scaled to a reference temperature of 25°C using activa-
occur along the elevation gradient (Asner etal., 2014a; Girardin tion energies of 64.8 and 37.0kJmol(cid:3)1 for V and J ,
cmax max
etal.,2014b;Silman,2014),reflectinglocalgeologicalsubstrates, respectively (Farquhar etal., 1980). Finally, rates of A
as well as changes in growth temperature, cloud cover and light obtained at ambient CO concentrations of 400 and
2
environment.Inadditiontomarkedinter-sitedifferencesintotal 2000lmolmol(cid:3)1 (A and A , respectively) were extracted
400 2000
soil [N] (0.6–15.5gNkg(cid:3)1), substantial variation in total soil from the A↔C curves and reported separately.
i
[P] occurs across both the lowland (38–727mgPkg(cid:3)1) and As atmospheric CO was not always saturating for measure-
2
upland sites (496–1631mgPkg(cid:3)1) (Table1). Soils at three of mentsofuplandspecies(becauseoflowatmosphericpartialpres-
the lowland sites in northern Peru (JEN-12, ALP-30 and ALP- sure, resulting in insufficient CO -saturated rates of A to enable
2
40) are notable for being low nutrient status arenosols/podzols calculationofJ ),itwaslikely thatJ mayhavebeenunder-
max max
(‘white sands’). Among the lowland and upland sites, mean estimatedin some cases; where this was likely to be the case (i.e.
annual precipitation (MAP) values range from 1560 to where there was no clear plateauing of A at high C values), we
i
5300mmyr(cid:3)1. Mean annual temperature ranged from 8.0 to excludedtheresultantJ valuesfromtheAndeandataset.With
max
18.8°C across the upland sites, and 24.4 to 26.6°C among the the exception of a few cases (e.g. Schefflera sp.; Fig.1), A↔C
i
lowlandsites. curvestypicallyflattenedoutathighC values(>90%ofcurves),
i
At each site, tree climbers collected upper canopy branches with A increasing slightly as C values increased further (see
i
(supportingleavesconsideredtobetypicallyexposedtofullsun- Fig.1), suggesting that feedback inhibition of A through limita-
lightformuchoftheday)fromdominanttreespecies.Therewas tionsintriose-phosphateutilization(TPU)wasunlikely.
little replication of individual species possible at any site. Each
treewasinitiallyidentifiedtothegenusleveland,wheneverpossi-
Leafstructureandchemistrydetermination
ble, to the species level. A total of 353 individual trees drawn
from 210 species were sampled across the 18 sites. See Support- Leaves were collected immediately following the gas exchange
ingInformationMethodsS1forfurtherdetails. measurements.Initially,theleafmidribwasremoved;thereafter,
a digital photograph was taken using a high-resolution scanner
(CanoScan LiDE 210; Canon, Hanoi, Vietnam) and later anal-
Leafgasexchangemeasurements
ysed for leaf area (IMAGEJ, v.1.38; National Institutes of Health
Measurements of leaf gas exchange were made during July to (NIH),USA).Leaveswerethenplacedinanovenat70°Cforat
September 2011, using portable photosynthesis systems (Li-Cor least48h,thedry masswasmeasuredandtheleafmassperunit
6400XTinfraredgasanalyser;Li-Cor BioSciences, Lincoln,NE, leaf area (M ) was calculated. Total leaf N and P concentrations
a
USA). Measurements were made on the most recently fully in dried leaves were determined using the Kjeldahl acid digest
expandedleavesattachedtothecutbranches(whichhadbeenre- method,asdetailedinAyubetal.(2011).
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2)
DescriptionofthesampledPeruvianfieldsitesTable1 TotalsoilnutrientsLeafchemistryAtm.[P]LeafNLeafPSiteElevationNo.ofMATMAPPressureSoil[N]aa(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)°1122)(mgkg)(gm)(gm)LeafN:PM(gmCategorycodeLatitudeLongitude(masl)species(C)(m)(kPa)classification(gkga (cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)LowlandSUC-053.255872.89421322026.22.75100Alisols1.92761.940.610.060.0430.17.0312931(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)TAM-0512.830969.2705223824.41.9099Cambisols1.62562.140.270.080.0228.69.4911927(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)JEN-114.878173.62951311826.62.70100Acrisols1.81412.120.520.060.0227.910.414437(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)ALP-013.950073.43331201825.22.69100Gleysols0.61101.900.400.080.0326.28.6211924(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)SUC-013.251972.90781171726.22.75100Plinthosols1.73051.810.630.090.0322.14.9912327(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)JEN-124.899073.62761351926.62.70100Podzols6.91331.970.520.090.0521.910.4215631(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)ALP-303.954373.42671502125.22.69100Arenosols0.8381.670.470.090.0420.86.8514546(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)CUZ-0312.534469.05392051224.41.9099Cambisols2.47271.880.470.100.0417.25.9710918(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)ALP-403.941073.44001421226.32.76100Podzols2.1591.840.360.100.0216.85.0017150(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)TAM-0912.830969.28432191324.41.9099Alisols1.13262.190.450.140.0316.43.7710521(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)TAM-0612.838569.29602151324.41.9099Alisols1.75292.560.340.170.0415.32.8412626 (cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)UplandSPD-0213.049171.536515271918.85.3083Cambisols8.816312.230.450.160.0515.44.0512636(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)SPD-0113.047571.542317762117.45.3085Cambisols11.910712.250.350.160.0414.33.3412429(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)TRU-0813.070271.555918852018.02.4782Cambisols8.14961.990.360.120.0516.93.5416538(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)ESP-0113.175171.594828631713.11.5672Umbrisols14.89812.390.500.190.0512.71.7814032(cid:4)(cid:4)(cid:4)(cid:3)(cid:3)(cid:4)0.440.210.0410.52.3516440TRU-0313.109771.599530441311.81.7871Umbrisols15.57872.24(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)WAQ-0113.190871.587430451311.81.5672Umbrisols8.814142.680.420.240.0511.52.1614946(cid:3)(cid:3)(cid:4)(cid:4)(cid:4)(cid:4)TRU-0113.113671.60693379168.01.9867Umbrisols15.08562.530.310.210.0411.23.1015149 Lowlandsitesarelistedinorderofdecreasingleafnitrogen(N):phosphorus(P)ratios,whileuplandsitesarelistedinorderofincreasingelevation.ExtremelylowsoilPdidnotnecessarilyproducelowleafPasinthecaseofALP-03andALP-04,andthereforelowlandsiteswererankedaccordingtotheleafNtoPratio,whichprovidesabetterindicationofnutrientlimitation(Aerts&Chapin,2000).AtmosphericpressurewasobtainedfromaLi-Cor6400XTgasexchangesystem.Foreachsite,asitecodeisshownasdesignatedbytheJointAmazonCarnegieRAINFORExpedition(JACARE);valuesoftotalsoilNandPareshown(expressedperunitsoildrymass).Alsoshownareaverageleafarea-basedconcentrationsoftotalnitrogen(N)andphosphorus(P),aswellastheaa),allshownwithSD.SoilclassificationfollowstheWorldReferenceBase(WRB).asl,abovesealevel;MAP,meanannualprecipitation;MAT,meanratioofleafN:Pandleafmassperunitarea(Maannualtemperature.Sources:Asneretal.(2014a),Quesadaetal.(2010;pers.comm.)andY.Malhi(unpublished).
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60 (see Methods S3 for details of optimization of protein assays).
(a) Frozensamplesof0.50cm2weregroundinEppendorftubesand
washed consecutively in 100% methanol, hexane and acetone.
45 Treated leaf powder was then resuspended in protein extraction
)–1 buffer (140mM Tris base, 105mM Tris–HCl, 0.5mM
s
–2 ethylenediaminetetraacetic acid, 2% lithium dodecyl sulfate
m
30
ol Cecropiaangustifolia (LDS) and 10% glycerol) containing 5mM DTT and protease
m
inhibitor cocktail (Sigma-Aldrich, Castle Hill, NSW, Aus-
µ
A( Glycydendronamazonicum tralia), heated for 10min at 100°C to completely dissolve
15
extracted protein, then clarified by centrifugation (14000g;
10min; room temperature). The supernatant was tested for
proteincontent.
0
Equivalent volumes of supernatant were diluted in 49 SDS-
60
PAGE sample buffer (Invitrogen Life Technologies, Carlsbad,
(b)
CA,USA)thenloadedontogels.Asweextractedproteinfroma
known amount of leaf area, we were able to analyse our samples
45
) 1 on an equivalent leaf area basis. Varying concentrations of
–
s Rubisco, purified from tobacco (Nicotiana tabacum), were also
2
–
m 30 Citronella incarum loadedontogels,servingasacalibrationseries.Proteinswererun
mol on4–12%NuPAGEBis-Trisgels(InvitrogenLifeTechnologies)
µ according to the manufacturer’s instructions and transferred to
(
A 15 Schefflera sp. Immobilon-P PVDF membranes (Merck Millipore, Kilsyth,
Vic., Australia) using an XCell II Blot module (Invitrogen).
Membranes were blocked with 5% skimmed milk powder in
0
0 20 40 60 80 100 120 140 Tris-bufferedsalinecontaining0.5%Tween-20(TBS-T)andan
C (Pa) antibody raised in rabbits against tobacco Rubisco (used at
i
1:5000)preparedbySpencerWhitney(ResearchSchoolofBiol-
Fig.1 FittedcurvesoftheresponseofCO2assimilationrate(A(area- ogy, Australian National University, Canberra). Secondary anti-
based))tointercellularCO (C)atsaturatinglightfor(a)alowlandspecies
2 i body (goat-anti-rabbit-alkaline phosphatase conjugate; Agrisera,
Glycydendronamazonicum(TAM-09)andanuplandspeciesCecropia
Vannas, Sweden) was diluted 1:5000. Blots were visualized
angustifolia(SPD-01)and(b)twouplandspecies,Citronellaincarum
(TRU-03)andSchefflerasp.(WAQ-01).Closedcirclesarethemeasured using the Attophos AP fluorescent substrate system (Promega,
ratesofassimilation,A.Solidlinescorrespondtofittedresponseand Madison, WI, USA) and imaged using a Versa-Doc (Bio-Rad,
dashedlinescorrespondtoestimatedresponseathighCi.Themaximum Hercules, CA, USA) imaging system. Blots were analysed using
Rubiscocarboxylationcapacity(V )wascalculatedfromthecurvature
cmax QUANTITYONEsoftware(Bio-Rad)andtherelativebanddensities
ofthedashedlineandthemaximumelectrontransportrate(J )was
max of each protein were determined from duplicate samples, and
calculatedfromthepointswhereAsaturated.Individualleaveswere
measuredattemperaturessetclosetotheprevailinggrowthtemperature, data averaged. The Rubisco concentration was calculated from
andthereforeV andJ werethennormalizedto25°C.CO wasnot the large subunit (molecular mass of 55kD and 16% N by
cmax max 2
alwayssaturatingformostuplandmeasurementsbecauseoflowpartial weight).
pressureand/orphosphatelimitation.
EstimationofNallocationinphotosyntheticmetabolism
ChlorophyllandRubiscomeasurements
N allocation in three major photosynthetic components (pig-
Leaf discs from mature leaves adjacent to the gas exchange leaf ment–protein complexes, electron transport and Rubisco) for all
were collected and transferred to a (cid:3)80°C cryogenic field con- leaves was estimated from chlorophyll concentration, V and
cmax
tainerforsubsequentchlorophyllandRubiscoassaysinthelabo- J , respectively. N allocation to pigment–protein complexes
max
ratory. (n )wascalculatedbyassuming44molNpermolofchlorophyll
P
The chlorophyll content of each set of leaf discs was deter- (Evans, 1989). N allocation to Rubisco (n ) was estimated from
R
mined using a dual-beam scanning UV-VIS spectrometer values of V according to Harrison etal. (2009), with slight
cmax
(Lambda 25; Perkin-Elmer, Shelton, CT, USA) after extraction modification (2.33mol CO (mol Rubisco sites)(cid:3)1s(cid:3)1 for the
2
of chlorophyll pigments from two frozen leaf discs (0.77cm2 catalytic turnover number of Rubisco at 25°C; Harrison etal.,
each)with100%acetoneandMgCO ,asoutlinedinAsneretal. 2009).WehereassumedthatallRubiscowasfullyactivatedand
3
(2014b). Chlorophyll a:b ratios varied between 2.45 and 2.75, mesophyllconductancewasinfinite.TheallocationofNtoelec-
whichisconsistentwithresultsofpaststudiesontropicaltreesin tron transportcomponents (n )wascalculatedfromJ assum-
E max
thePeruvianAmazon(Asner&Martin,2011). ing 160mol electrons (mol cytochrome f )(cid:3)1 s(cid:3)1 and 8.85mol
Protein was extracted from frozen leaf discs following the N(mmolcytochromef )(cid:3)1(Evans&Seemann,1989).Thepro-
methodoutlinedinGasparetal.(1997)withslightmodifications portion of total leaf N allocated to each photosynthetic
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component was calculated by dividing the N investment in each (r2=0.37; P<0.01) and elevation (r2=0.48; P<0.01). Weaker
componentbythetotalNcontentperunitleafarea. positive associations were observed for area-based leaf [N] (N )
a
with total soil [N] (r2=0.10; P<0.01) and elevation (r2=0.14;
P<0.01).
Dataanalysis
Leaf mass per unit leaf area (M ) varied widely, both among
a
Log transformations were carried out on leaf trait values when andwithinlowland(54–230gm(cid:3)2)andupland(60–249gm(cid:3)2)
10
necessary to ensure normality and minimize heterogeneity of sites (Tables1, S1). Although variations in M were not corre-
a
residuals.Studentt-tests(two-tailed)wereusedtocompareover- latedwithvariationsinsoil[P],thereweresignificant(butweak)
allmeansoflowlandanduplandspecies.Standardizedmajoraxis correlations between M and total soil [N] (r2=0.04; P<0.01)
a
(SMA) estimation was used to describe the best-fit relationship and elevation (r2=0.03; P<0.01) (Table S2). The overall mean
betweenpairsofvariablesandtoassesswhetherrelationshipsdif- of M for the sampled upland species (143(cid:4)39gm(cid:3)2) was sig-
a
fered betweenlowland and upland elevationclasses, using SMATR nificantly higher than that of the lowland species
version 2.0 software (Falster etal., 2006; Warton etal., 2006). (132(cid:4)35gm(cid:3)2;Table2,P<0.05).
The decision to compare upland and lowland trait relationships Across all 18 sites, leaf N was positively correlated with M
a a
reflectsthestrongelevationcontrastinenvironments,phylogeny, (P<0.01; r2=0.12; Table S2), with the N ↔M relationship
a a
floristic composition and forest structure (Gentry, 1988; van de being stronger among upland than lowland sites (r2=0.07 for
Weg etal., 2009; Asner etal., 2014b). The significance of SMA lowlandsitesandr2=0.20forupland;seeTableS3forP-values,
regressionwastestedata=0.05. slopes and intercepts of each SMA relationship). The slope and
In addition to the above bivariate analyses, we also used a intercept of the relationship differed between the two elevation
mixed-effectslinearmodelcombiningfixedandrandomcompo- classes (Fig.2a) – upland species exhibited higher N for a given
a
nents(Pinheiro&Bates,2000)toaccountforvariabilityinarea- M than lowland species, particularly in low M species. Across
a a
and N-based rates of V , and area-based rates of J . This all sites, leaf P exhibited a weak, positive correlation with M
cmax max a a
approach enabled thestructured nature of the data set to be rec- (P<0.01; r2=0.04; Table S2). Similarly, a weak positive
ognized, and for interactions between multiple terms to be con- P ↔M relationship(P=0.003;r2=0.04;TableS3)wasfound
a a
sidered. The model’s fixed effect included continuous amonguplandspecies(Fig2b).AlthoughnosignificantP ↔M
a a
explanatory variables only: leaf traits (M , and area-based leaf N relationshipwasfoundamonglowlandspecies(withleafP vary-
a a
andP),andenvironmentvariables(soil PandNconcentrations, ing 20-fold; Table S1), mean values of P at a given M were
a a
mean annual temperature (MAT) and the effective cation lowerthanthoseoftheiruplandcounterparts.
exchangecapacityofsoil(ECEC)).Modelspecificationandvali-
dationwerebasedontheprotocolsoutlinedinZuuretal.(2009)
Variationsinphotosyntheticmetabolism
and fitted using the nlme package (R package v.3.1-105; R
FoundationforStatisticalComputing,Vienna,Austria;RDevel- Light-saturated rates of photosynthesis per unit leaf area, mea-
opment Core Team, 2011). Details of the model selection pro- sured at the prevailing daytime air temperature (T) at each site
cess are provided in Table S6. Briefly, phylogeny (family/genus/ and at an atmospheric CO concentration of 400lmolmol(cid:3)1
2
species) was treated as a nested random effect, placing focus on (A ), differed among co-occurring species (Table S1). How-
400,a
thevariationcontainedwithinthesetaxonomicterms,ratherthan ever, there was no significant difference between mean values of
meanvaluesforeachlevel.Sitevariationwascapturedbysoiland A fromlowlandanduplandclasses(Table2).Thisuniformity
400,a
environmental factors considered in the model’s fixed compo- of A occurred despite significantly lower measuring T values
400,a
nent; because of this, no site term was included in the random at the high-elevation sites (overall means: lowland 29.4(cid:4)0.9°C;
component. Model comparisons and the significance of fixed- upland25.7(cid:4)2.1°C;P<0.05)andlowerintercellularCO par-
2
effects terms were assessed using Akaike’s information criterion tial pressure (C) (overall means: lowland 28.4(cid:4)3.7Pa; upland
i
(AIC). Unless otherwisestated, statistical analysiswasperformed 18.8(cid:4)3.0Pa;P<0.05)(TableS4).AssessedonaperunitleafN
usingSPSSv.20(IBMCorporation,Armonk,NY,USA). basis (A400,N), average rates were lower at the upland sites com-
pared with their lowland counterparts (Tables2, S4), reflecting
higher leaf N for trees at high elevation (Table1). Across sites,
Results a
mean A decreased with decreasing MAT (Fig. S1d). Area-
400,N
based rates of photosynthesis at elevated CO (A ) were
Variationsinleafchemistryandstructure higher in upland (17.1–26.5lmolm(cid:3)2s(cid:3)1; Tab2le S42)00t0h,aan low-
Among lowland sites, there was a six-fold variation in leaf N:P land (16.1–22.6lmolm(cid:3)2s(cid:3)1) species (P<0.05). The higher
ratios (7.6–45.9) (Table S1), but for upland sites, when ranked values of A at the upland sites were achieved despite the
2000,a
according to increasing elevation, mean values of leaf N:P were coldertemperatures.OnaperunitleafNbasis(A ),average
2000,N
largelyconsistentacrosssitesofsimilarelevation(Table1).Across rates were similar for the two elevation classifications (Table S4;
all sites (lowland and upland combined), variations in leaf N:P Fig.S1e).
ratios were predominantly driven by variations in leaf [P] To explore differences in the rates of the underlying compo-
(r2=0.59;P<0.01;TableS2)ratherthanleaf[N].Variationsin nents of net photosynthesis, we compared maximal area-based
area-based leaf [P] (P ) were positively correlated with soil [P] rates of CO fixation by Rubisco (V ) and photosynthetic
a 2 cmax,a
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MeanvaluesandstandarddeviationofleaftraitsforuplandandlowlandspeciesTable2 252525VAAVJ;;;cmaxamaxacmaxN400,a400,Nlllll25Leaf(mol(mol(mol(mol:(molLeafNLeafPLeafMJ;maxaaaa(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)(cid:3)252222111212111traitsmgNmmVgN(gm)(gm)N:P(gm)s)s)s)s)s)nnnn;cmaxaAPRE (cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)aaaaaaaaaaaaaaLowland1.960.520.090.0522.28.6132358.23.94.32.235.914.666.718.61.860.4018.98.10.370.110.240.090.090.040.030.01species(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)(cid:4)bbbbabbbabaabbUpland2.310.440.180.0613.53.6143397.63.63.41.748.820.096.936.91.920.3622.59.40.380.080.220.070.110.040.030.01species Valuesareexpressedonanareabasis.LeafN,leafnitrogen;leafP,leafphosphorus;leafN:P,leafnitrogentophosphorusratio;M,leafmassperunitleafarea;A,area-basedlight-saturatedaaa400,a(cid:3)(cid:3)ll11netphotosynthesismeasuredat400molmolatmospheric[CO];A,area-basedlight-saturatednetphotosynthesismeasuredat400molmolatmospheric[CO]perunitleafnitrogen;2400,N2°°25252525VmaximumcarboxylationvelocityofRubisconormalizedto25C;Jmaximumrateofelectrontransportnormalizedto25C;J:V,ratioofmaximumRubiscocarboxyla-;;;;cmaxamaxamaxacmaxa,,°°25tionvelocityovermaximumrateofelectrontransport,bothnormalizedto25C;V,ratioofmaximumcarboxylationvelocityofRubisconormalizedto25CperunitleafN;n,totalfraction;cmaxNA–,fractionofleafNinpigmentproteincomplexes;n,fractionofleafNinRubisco;andn,fractionofleafNinelectrontransport.Valuesareover-ofleafNallocatedtophotosyntheticmetabolism;nPRE(cid:4)<allmeanSDofleaftraitsforlowlandanduplandsites.Significantlydifferentmeansareindicatedbydifferentletters(P0.05). DurpeFrlloIvuiTwerplNIaTtoonaneehem2–llilP(g m) aphnpaawwgeeharlavlfs–2(g mN)g-elalolraotllcvpaysbe.uuputyatllaaastraat2naaiieletspgomwmhns,orcennpltdeec0000s,eohrrnirdLtddohmat....0124eodetteSoshn0016sowitieoa....arpthnsseevnr50001464i1egspnaoencpptfuehfiteitp-)ergilnfise5eesctrtlmpelta,dssrVonylrh0iccarcs(n’eTsyaae(ihii(Pgb,ientiesceefisfaarb,tnpeahm)ei)faisssarupcb)agn).mrnecte;;eapan(aamellswrhSroogexVcodlnrnossaee;oettyntlapaeeaeVetSatchrnisant2tsiseenmtgsto3ndohceih5soceinn(gthvwm.daifnedPee(arffxiueatlfdssJsnsadaate;ao(t2eiyharm)pxanrdrnswdr2rolm5,iie()ia,ialNeipae5dnningvfVae°lzxbtlnlsbaeeine,asCrdloclacanotaaa,eJoadCtem)deuhntdlrfmnwuil,wsaseaoamei(wn,cpd2acaxtiluiwnlxsouii;tl(l.ana5lhaotiaa;isehaebspgartmhvjd2nnnJi°eooa.shh2ilny5neemdaCstdn5frgutiim,nirVe;gnfioanpMvaepasarxcdTccwepaxncJs-(log;oldvaomaaoemasVliaesenusaeg(2nawoinaumcacpNa,t(5brecel(dxftphixneegmuiSeesp;nT)l;mt(actasMeg steawaPh(av2emips2i.exs1resanr5r2iai5;<eairSAeeiest4NtsnMggr,–p;cnnMrea.ysa)9a0i2gn2ie.taSPaedopnt)a.5(eAVAolirM0eNbsNoernWfi)drt.3<sf5mcTirwwarsdatcAClmo6)es)uti↔i0J;aal(hagtlhfataanrmf(oTnrty.VhlxeeronFeena0isitea)ttMngarizdsfenhcdxwi,5lsnhelmeerbg;ydcesedasea)r(i-dao2cpi.laap2bo,gsksseatxs0omuhf5hsyeoS)inh;ren40tiNt)n2ampcioooeslm1oyde.e5Sgirt;fl2nlbetaaebauhra%eoP5oSpssvPares,afoopnlne):eabesiachealplt(rsydm<sr↔r)tvlngsMoeoo(,vihini.ladinonFaitlrv0eegiitatnSmotdMbpggegdia9h)iri.ngiso.0eniosh7lmeeyaligtt.suffn5nuietahhnoo3tireri)snpyeecrr---,..;,
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100 200
–2–1ms) 80 (a) –2–1ms) 150 (b)
mol 60 mol 100
25V(µcmax,a 2400 25(µJmax,a 50
0 0
3.5 50
25Vcmax,a 322...005 (c) –2–1s)mol m 3400 (d)
25:Jmax,a 11..50 25(µmax,N 1200
Vc
0.5 0
Fig.3 Boxandwhiskerplotsof(a)maximumcarboxylationvelocityofRubisconormalizedto25°C(Vcmax;a25),(b)maximumrateofelectrontransport
normalizedto25°C(Jmax;a25),(c)Jmax,25:Vcmax,25ratio,and(d)ratioofVcmax;a25overleafnitrogen(N)(Vcmax;N25)foreachsite.Valuesareexpressedonan
areabasis.SitesarearrangedaccordingtodecreasingleafN:phosphorus(P)forlowlandsitesandincreasingelevationforuplandsites.Theupperand
loweredgesofeachboxindicatethe75thand25thpercentiles,respectively.Thehorizontallinewithineachboxisthemedianandtheverticalbarsindicate
the10thtothe90thpercentileranges.
ratios were observed among individuals (Figs3, 4), underpinned
200
Citronella incarum byfundamentaldifferencesintheCO2responseofnetphotosyn-
thesis (e.g. Fig.1b). In most leaves, Jmax;a25 and Vcmax;a25 co-
varied, resulting in relatively constant Jmax;a25:Vcmax,25 ratios, as
150 Cecropia illustratedbydatafromindividualplants ofCecropiaangustifolia
aungus(cid:3)folia
–1s) and Glycydendron amazonicum where the Jmax;a25:Vcmax;a25 ratio
2 was 1.8 (Figs1a, 4). However, some leaves exhibited high
–m
µmol 100 Gamlyacyzdoennicdurmon wVchmearxe;a2J5mabx;ua2t5l:oVwcmJamx;aax2;5a25=(1F.1ig).w1bh;ileinodtihveidruleaalvoesf wScihthefflaesriamsipla.r,
25(max,a iVnccmaarxu;am25 hinaddivmidaurakledliyn hiFghige.r1bJ)max;ale2a5di(neg.g. ttohe aCitrhoingehlelar
J 50 Schefflerasp. Jmax;a25:Vcmax;a25value (2.4). Such variations in Jmax;a25 and
Vcmax;a25 probablyreflectintra-and/orinter-specificvariationsin
relative allocation of N allocation to Rubisco vs electron trans-
Lowland Upland port/bioenergetics.
0
0 20 40 60 80 100
Bivariaterelationships
V 25(µmol m–2s–1)
cmax,a
Fig.4 PlotofmaximumcarboxylationvelocityofRubisconormalizedto Across all18sites,Vcmax;a25 andJmax;a25 exhibitedpositive corre-
25°C(Vcmax;a25)againstmaximumrateofelectrontransportnormalizedto lationswithsoilP,soilNandelevation,andnegativecorrelations
25°C(Jmax;a25).Datapointsrepresentindividualleafvalues(138lowland with MAT (Table S2); the strength of these relationships was
sdpeepcicietsedanindt6h9euAp↔lanCdscpuercvieess)i.nAFrirgo.w1.sCcolorsreedspsoynmdbtoolst,hleowfoluanrdspsepceiecsies; greater for Jmax;a25 than Vcmax;a25. Relationships with MAP were
opensymbols,uplandispecies. either weak (Jmax;a25) or not significant (Vcmax;a25) (Table S2).
Acrossallsites,variationsinVcmax;a25andJmax;a25werealsocorre-
lated with leaf chemical composition traits (Table S2), with
was particularly pronounced at the upland sites (Fig.3; bivariaterelationshipsbeingstrongerwithP (P<0.01;r2=0.11
a
TableS1). for Vcmax;a25; r2=0.13 for Jmax;a25) than Na (P<0.01; r2=0.05
Variations in Jmax;a25 were strongly correlated with Vcmax;a25, for both Vcmax;a25 and Jmax;a25). Leaf N:P ratios exhibited weak,
bothforlowland(r2=0.59)anduplandclassifications(r2=0.75) negative correlations with Vcmax;a25 and Jmax;a25 (P<0.01;
(Fig.4). Overall, the Jmax;a25↔Vcmax;a25relationship was similar r2=0.08forVcmax;a25,r2=0.06forJmax;a25;TableS2).Nosignif-
in the two elevation groups, with mean Jmax;a25:Vcmax;a25 ratios icant relationship was found between Vcmax;a25 and Ma, whereas
being statistically equivalent in lowland and upland classes the Jmax;a25↔Ma relationship was significant (P<0.05;
(Table2). Importantly, marked differences in Jmax;a25:Vcmax;a25 r2=0.04;TableS2).
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(a) (b) (c) (d)
1–s) 80
2–m
ol
m
µ
( 20
25max,a
Vc
5
240
(e) (f) (g) (h)
–1s)
2
–m
mol 60
µ
(
25max,a
J
15
50 200 0.5 1.0 2.0 4.0 0.01 0.05 0.50 4 16 64
Ma(g m–2) Na(g m–2) Pa(g m–2) Leaf N:P
Fig.5 Upperpanels:log-logplotsofmaximumcarboxylationvelocityofRubisconormalizedto25°C(Vcmax;a25)inrelationto(a)leafmassperunitleaf
area(M),(b)leafnitrogen(N)area(N),(c)leafphosphorus(P)area(P)and(d)leafN:P.Datapointsrepresentindividualleafvalues(150lowland
a a a
speciesand95uplandspecies).Standardizedmajoraxis(SMA)testsforcommonslopesrevealedsignificantdifferenceswhencomparingVcmax;a25↔Na,
Vcmax;a25↔PaandVcmax;a25↔leafN:Prelationshipsbetweenlowlandanduplandspecies,butnosignificantdifferencewhencomparingslopesof
Vcmax;a25↔Marelationshipsbetweenlowlandanduplandspecies.Lowerpanels:log-logplotsofmaximumrateofelectrontransportnormalisedto25°C
(Jmax;a25)inrelationto(e)Ma,(f)Na,(g)Paand(h)leafN:P.Datapointsrepresentindividualleafvalues(127lowlandspeciesand58uplandspecies).
SMAtestsforcommonslopesrevealedsignificantdifferencewhencomparingJmax;a25andleaftraitsrelationshipsbetweenlowlandanduplandspecies.
Closedsymbols,lowlandspecies;opensymbols,uplandspecies.SMAregressionsaregivenonlywhentherelationshipsaresignificant(P<0.05);referto
SupportingInformationTableS3.
Whenassessedamonguplandsites,nosignificantrelationships opposed to N (and vice versa) (Gu€sewell, 2004). No significant
werefoundbetweenVcmax;a25,Ma,Na,PaorN:Pratio(Fig.5a– difference in the slopes of the relationships was found (P>0.05;
d). For lowland sites, Vcmax;a25 was positively related to Pa data not shown). Similar patterns were observed for Jmax;a25
(P=0.013; r2=0.04; Table S3) and N (P=0.050; r2=0.02; (Fig.5e–h), which was positively related to N (P=0.012;
a a
TableS3),butnotleafN:PratioorM (Fig5a–d).Theabsence r2=0.05; Table S3) and P (P=0.002; r2=0.08; Table S3) for
a a
of an N:P effect for upland or lowland classes was consistent thelowlandclassonly.
with SMA analyses comparing the slopes of Vcmax;a25↔Na, Investigating whether variations in photosynthetic N-use effi-
Vcmax;a25↔Pa and Vcmax;a25↔Ma for the lowland class, split ciency were related to Ma, both across all sites (Table S2) and
accordingtoleafN:Pratiosbelowandabove20–thisratiogen- within each elevation class (Fig.6a), there was no significant
erally being thought indicative of the N:P ratio above which Vcmax;N25↔Ma relationship across all 18 sites (Table S2) or
physiological processes are more likely to be limited by P as within the upland elevation class (Table S3). Nevertheless, for
80
1––1Ns) 40 (a) (b) (c)
ol g 20
m
µ 10
(
25max,N 5
Vc
2.5
50 200 0.01 0.05 0.50 4 16 64
Ma(g m–2) Pa(g m–2) Leaf N : P
Fig.6 Log–logplotsoftheratioofmaximumcarboxylationvelocityofRubisconormalizedto25°C(Vcmax;a25)toleafnitrogen(N)(Vcmax;N25)inrelationto
(a)leafmassperunitleafarea(M),(b)leafphosphorus(P)area(P)and(c)leafN:P.Datapointsrepresentindividualleafvalues(150lowlandspecies
a a
and95uplandspecies).Standardizedmajoraxis(SMA)testsforcommonslopesrevealedasignificantdifferenceonlywhencomparingVcmax;N25↔Pa
betweenlowlandanduplandspecies.Symbols:closedsymbols,lowlandspecies;opensymbols,uplandspecies.SMAregressionsaregivenonlywhenthe
relationshipsaresignificant(P<0.05);refertoSupportingInformationTableS3.
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the lowland class, a weak negative Vcmax;N25↔Ma relationship There was considerable variability in nA among lowland and
was observed (P=0.01; Table S3). On average, Vcmax;N25 at a upland species (0.1–0.6), with significant negative correlations
given M was higher in upland species than in their lowland being found with M , N and P for the lowland group (Fig.8;
a a a a
counterparts. With respect to foliar P, there was no significant Table S5). Similar significant correlations existed for the upland
relationship between Vcmax;N25 and leaf Pa or leaf N:P ratio class but with the important caveat that upland species consis-
whenconsideringtheelevationclassesseparately.Thisconclusion tently exhibited higher n at a given N and P (Figs8, S2;
A a a
held for Vcmax;N25↔Pa when combining upland and lowland TableS5).Thus,whilemeanvaluesofnAweresimilarinupland
data (Table S2). For Vcmax;N25↔ N:P, combining upland and andlowlandspecies,thefractionofleafNallocatedtophotosyn-
lowlanddataresultedinaweaksignificantrelationship(P<0.05; thesiswasgreaterinuplandplantswhencomparisonsweremade
r2=0.02; Table S2); similarly, relationships between Vcmax;N25 atcommonleafNaandPavalues.
and soil P, soil N and elevation were relatively weak (Table S2).
Collectively, these results show that the proportion of the vari-
ValidationofRubiscoestimatesbyinvitroassays
ance in Vcmax;N25 accounted for by the above soil- and leaf-level
parameterswasnegligible. We used in vitro Rubisco assays on 16 lowland species (Fig.9a)
to quantify n , thus allowing direct comparison with that of the
R
VariationinN-allocationpatterns in vivo estimates derived from Vcmax;a25. Fig.9(b) shows that
there was considerable discrepancy between in vitro and in vivo
Tofurtherexplorewhatfactorsmightcontributetovariationsin predicted n . If one assumes that the in vitro values provide an
R
Vcmax;N25,wecalculatedthefractionofleafNallocatedtophoto- estimateofpotentialRubiscocapacity,andthattheinvivovalues
synthesis (n ); n is dependent on the allocation of leaf N to areindicativeoftherealizedmaximumrateinintacttissues,then
A A
Rubisco (n ),electron transport(n )andpigment–proteincom- itispossiblethattheinvivoapproachunderestimatesthepropor-
R E
plexes (n ). Fig.7 shows thatmean values of n andits underly- tion of N allocated in Rubisco. Reliance on the in vitro values
P A
ing components exhibited relatively little variation across sites. resultedinmarkedincreasesinn atagivenM ,albeittheoverall
R a
Nevertheless, inter-specific variations were evident at each site, pattern of increasing n with decreasing M still held (Fig. S3a).
R a
with n varying up to seven-fold at some sites (e.g. CUZ-03; Considering the overall N investment pattern in photosynthetic
R
0.03–0.20;TableS1).AlargeproportionofNwasinferredtobe metabolism,adoptinginvitroestimatesofn resultedinmarked
R
allocated to pigment–protein complexes, with n being greater increases in the total fraction of N allocated to photosynthesis
P
thann andn combined.Theoverallmeanofn fortheupland comparedwithinvivoestimates(Fig.S4).Indeed,insomecases
R E R
class (0.105) was significantly higher than that for the lowland in vitro estimates of N allocation to Rubisco was similar to, or
class (0.090; Table2; P<0.05). Similarly, n was higher for even higher than, N allocation to pigment(cid:3)protein complexes
E
upland (0.034) than for lowland groups (0.028; Table2; (Fig.S4).Collectively,theseresultssuggestthattheanswertothe
P<0.05). There was no difference between the elevation classes question ‘How much leaf N is allocated to photosynthesis’ will
in n . Overall, n was similar in the lowland and upland group- dependonwhetherinvivoorinvitroestimatesofn areusedin
P A R
ings(37–38%;Table2). theunderlyingcalculations.
0.6
n
P
n Lowland sites Upland sites
E
0.5
n
n nts R
ated ipone 0.4
cm
of N allohetic co 0.3
ction osynt 0.2
Frahot
p
0.1
0
Fig.7 Stackedgraphshowingthefractionofleafnitrogen(N)inpigment–proteincomplexes(n ),inelectrontransport(n),andtheinRubisco(n )for
P E R
eachsite.nRwasestimatedfromthemaximumcarboxylationvelocityofRubisco(normalizedto25°C),Vcmax;a25;nEwasestimatedfromthemaximum
electrontransportrate(normalizedto25°C),Jmax;a25;andnPwasestimatedfromthechlorophyllconcentration.nPvalueswereunavailableforsixsitesasa
consequenceofthawingofleafsamples.SitesarearrangedaccordingtodecreasingleafN:Pforlowlandsitesandincreasingelevationforuplandsites.
Errorbars,(cid:4)SEofmean.
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